Although Tuberculosis is a disease, for which we have a vaccine and sterilizing chemotherapy, WHO reported 8 million new cases and 1.6 million deaths from TB in 2016. This is surprising since the causative agent; Mycobacterium tuberculosis was isolated in 1882 by Robert Koch. When active TB develops, a cure normally requires six months of treatment, known as ?short-course? chemotherapy. However, for individuals infected with multidrug-resistant M. tuberculosis, the minimal duration of treatment is one to two years. We hypothesize the requirement for long treatment periods are the result of persistence --- the capacity of M. tuberculosis to resist sterilization in a mammal. Clearly, knowledge of persistence could lead to new strategies to control TB globally. However, the acquisition of basic M. tuberculosis knowledge was historically limited by the inability to transfer genes into this bacterium. This proposal was originally funded following our 1987 publication demonstrating that it was possible to transfer genes into M. tuberculosis using a mycobacteriophage cosmid vector (shuttle phasmid). Shuttle phasmids allowed for the development of genetic tools, including the first plasmid transformation system, efficient transposon mutagenesis, specialized transduction, and luciferase reporter mycobacteriophages for rapidly assessing drug susceptibilities. This resubmission for competitive renewal builds on our expertise with mycobacteriophage-based tools and our extensive preliminary data to study persistence. Gene transfer elucidated the mechanisms of action and resistance to the front-line TB drug isoniazid (INH), which led to our development of an in vitro model to observe INH-tolerant cells --- a subpopulation of M. tuberculosis cells (0.1 to 1 %) that survive INH exposure without resistance mutations. Importantly, we discovered that cysteine and vitamin C prevent INH tolerance, resulting in culture sterilization, and that INH tolerance is mediated by a stress-induced phenotype that correlates with slower cell division. We have further determined that inactivation of the non-essential sigma factor SigE leads to the loss of INH tolerance and that starvation for methionine and arginine also lead to sterilization of M. tuberculosis cultures. Moreover, our RNAseq analysis identified an island of M. tuberculosis genes that is activated by sterilizing conditions. This proposal plans to elucidate mechanisms of INH tolerance in vitro and in vivo using specialized transduction and libraries of barcoded deletion mutants. In addition, we will develop novel dual-reporter mycobacteriophages to visualize and quantitate persistent M. tuberculosis cells. Altogether, these new tools will enhance our understanding of persistence, thereby leading to improved TB therapies.